Reaction hijacking of tyrosine tRNA synthetase as a whole-of-life-cycle antimalarial strategy

Aminoacyl tRNA synthetases (aaRSs) are attractive drug targets. Here we show that class I and II aaRSs are previously unrecognized targets for AMP-mimicking nucleoside sulfamates. The target enzyme catalyzes the formation of an inhibitory amino acid-sulfamate conjugate, via a reaction-hijacking mechanism. We identified adenosine 5′-sulfamate (AMS) as a broad specificity compound that hijacks a range of aaRSs; and ML901 as a specific reagent that hijacks a single aaRSs in the malaria parasite, Plasmodium falciparum, namely, tyrosine RS (PfYRS). ML901 exerts whole-of-life-cycle killing activity with low nanomolar potency and single dose efficacy in a mouse model of malaria. X-ray crystallographic studies of plasmodium and human YRSs reveal differential flexibility of a loop over the catalytic site that underpins differential susceptibility to reaction-hijacking by ML901.

activity against bacteria (6)(7)(8), which lack E1 enzymes. The compounds are broadly toxic and have been reported to inhibit protein synthesis (8,9), but until now the mechanisms underlying these activities were unknown.
We explored the activity of adenosine 5′-sulfamate (AMS, Fig. 1B), a close mimic of AMP, as a potential starting point for identifying antimalarial compounds. We found that AMS is highly cytotoxic (IC 50_72h = 1.8 nM) to P. falciparum cultures, with an efficacy similar to that of current front-line drug, dihydroxyartemisinin (DHA), but is also cytotoxic to mammalian cell lines, such as HCT116 (IC 50_72h = 26 nM) (table S1). We found that treatment of P. falciparum cultures with AMS triggers eIF2α phosphorylation (Fig. 1C), a hallmark of stress caused by either accumulation of unfolded proteins or uncharged tRNAs (10). Like E1 enzymes, aminoacyl tRNA synthetases (aaRSs) are adenylate-forming enzymes (AFEs). They catalyze the transformation of amino acids into AMP conjugates, and then into aminoacyl-tRNAs, to supply protein synthesis. Given the reported effects on protein translation (8,9), we considered the possibility that aaRSs might be able to catalyze nucleophilic attack of AMS on their cognate aminoacyl tRNAs (Fig. 1A).
The proposed mechanism would be expected to generate AMS-amino acid conjugates (Fig. 1A), so we used targeted mass spectrometry to search for the predicted conjugates in P. falciparum-infected red blood cells (RBCs) and cultured human cells (HeLa) that had been treated with 10 μM AMS for 2-3 h (see Supplementary Material for full methods). Following Folch extraction of lysates, the aqueous phase was subjected to liquid chromatography-coupled mass spectrometry (LCMS) and the anticipated masses for the 20 possible amino acid conjugates were interrogated. In P. falciparum, the extracts yielded a strong signal for AMS-Tyr (Fig. 1D), with matching precursor ion m/z (< 3 ppm) and anticipated fragmentation spectrum ( fig. S2A). MS peaks were also detected for the adducts of Asn, Asp, Ser, Thr, Gly, Ala, Lys and Pro ( fig. S2B). In the mammalian cell line, AMS conjugates were identified for Asn, Pro, Ala, Thr, Asp and Tyr ( fig. S3). No peaks were detected in control samples. These data are consistent with aaRSs catalyzing nucleoside sulfamate attack on the activated oxy-ester bonds of their cognate aminoacyl tRNAs (Fig.  1A), Thus, both class I and class II aaRSs are potentially susceptible to inhibition via the reaction hijacking mechanism.

Identifying a nucleoside sulfamate with potent and specific antimalarial activity
In an effort to identify aaRS-targeting nucleoside sulfamates with narrower specificity, we screened 2314 sulfamates from the Takeda compound library (Cambridge, MA, USA) for inhibition of the growth of P. falciparum. The library included compounds that were synthesized as potential inhibitors of Atg7, an E1 that activates UBLs, including the Atg8s (11). We identified several pyrazolopyrimidine sulfamates with a 7-position substituent (exemplar ML901; Fig. 2A) that possess potent activity against P. falciparum. The ML901 50% inhibitory concentration (IC 50_72h = 2.0 ± 0.1 nM) is similar to that for DHA (table S1). ML901 was tested for cytotoxicity against different mammalian cell lines (table S1) and showed 800-to 5,000-fold selectivity towards P. falciparum (>1,000-fold higher selectivity than AMS). ML901 retained activity against all strains of P. falciparum tested, regardless of their resistance profile and geographical origin (table S2). It potently inhibited transmissible male gametes (table S2); and prevented development of P. falciparum in primary human hepatocytes (table S2). We confirmed that ML901 exerts activity against human Atg7 (IC 50 = 33 nM); but has much weaker activity against other E1 enzymes (table S3), as previously reported for nucleoside sulfamates with a substitution at the 7-position (11); and consistent with the low mammalian cell cytotoxicity. By contrast, AMS is a potent inhibitor of each of the E1s tested (table S3). The rat pharmacokinetic profile of ML901 ( fig. S4; table S3) is encouraging, with low blood clearance and a long terminal half-life in blood (T 1/2∞ = 41 h) following intravenous or oral dosing.
We determined the in vivo antimalarial efficacy of ML901 in severe combined immune deficient (SCID) mice, engrafted with P. falciparum infected human RBCs (12,13), which is the gold standard for testing in vivo efficacy of malaria drug candidates. A single dose

ML901 selectively targets plasmodium tyrosine tRNA synthetase
To identify the target of ML901 in P. falciparum, extracts of infected RBCs that had been treated with 3 μM ML901 (3 h) were subjected to LCMS to search for amino acid-ML901 conjugates. An LCMS peak corresponding to protonated ML901-Tyr (m/z = 576.1324) was detected. Synthetic ML901-Tyr was generated and spiked into the untreated P. falciparum

ML901 exerts its activity by hijacking the active site-bound reaction product
We examined the abilities of the recombinant YRSs to consume ATP, i.e., to form and release AMP-Tyr in the initial reaction phase. HsYRS and PfYRS S234C show higher activity (in the absence of tRNA) than PfYRS ( fig. S11B). This difference suggests that AMP-Tyr is bound less tightly to the Hs and mutant PfYRS active sites. Upon addition of the cognate tRNA Tyr , ATP consumption is enhanced, consistent with productive aminoacylation. Acylation of the cognate tRNA Tyr to radiolabeled tyrosine (19), occurs at a similar level in all three enzymes ( fig. S11C). ML901 inhibits ATP consumption by PfYRS when added in the presence of PftRNA Tyr but not in its absence ( fig. S11D). Similarly, ML901 inhibits tRNA Tyr acylation to tyrosine in vitro by PfYRS, but not by HsYRS (Fig. 4C). Synthetically generated ML901-Tyr conjugate is able to inhibit the activity of both PfYRS and HsYRS ( fig. S11E,F was subjected to LCMS analysis and we detected a peak at m/z 576.1331 Da (Fig. 4D,E), consistent with the expected protonated ML901-Tyr ion, and confirmed using the synthetic

A structured loop over the PfYRS active site facilitates reaction hijacking
To obtain crystals of ML901-Tyr-bound PfYRS, we incubated His-tagged PfYRS with tyrosine, ATP and ML901 in the presence of tRNA and then purified the complex using nickel affinity and gel filtration chromatography. The crystal structure (refined at 2.15-Å resolution) revealed a homodimer organization with clear density for the ML901-Tyr ligand (Fig. 5A, fig. S13A).
PfYRS is a Class I aaRS, characterized by a catalytic domain that adopts a Rossmann fold  . S15F), the 222 KMSSS 226 loops were not defined in the electron density ( Fig. 5Cii; fig. S15D,E). Moreover, His49 (the equivalent of PfYRS His70) adopts a position (Fig. 5D, magenta) similar to that in chain A of ML901-Tyr-bound PfYRS

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Europe PMC Funders Author Manuscripts (compare figures S13G,H and S15E), further suggesting that this configuration is associated with increased loop mobility. A comparison of the interaction networks ( fig. S13B-D, S15B,C) reveals notably fewer interactions with the ML901 moiety in HsYRS compared with PfYRS; and specific interactions were poorly conserved between the two enzymes.
We also solved the structure of PfYRS S234C in complex with synthetic ML901-Tyr ( fig.  S16). Similar to HsYRS, the KMSKS loops of both monomers were not defined in the electron density ( fig. S16E,F), and His70 adopts a rotamer that is not consistent with a structured KMSKS loop (Fig. 5Ciii,D, green; compare figures S13G,H and S16G,H). Potency and selectivity of ML901 for PfYRS thus appears to be associated with a stabilized loop over the active site. That is, the decreased susceptibility of HsYRS and PfYRS S234C to reaction hijacking by ML901 is associated with mobility of the KMSSS/KMSKS loop, which is, in turn, associated with rotation of the His49/70 side chain. These conformational changes may promote dissociation of the charged tRNA, thereby preventing the hijacking reaction.
The pyrazolopyrimidine sulfamate chemotype is an attractive starting point for a malaria drug discovery program, based on our observation that the specific inhibition of PfYRS by ML901 is lethal to disease-causing and transmissible stages of P. falciparum, and that ML901 exhibits a long in vivo half-life, underpinning its single-dose efficacy in a murine model of human malaria. Further exploration of substitutions at the 7-position of the pyrazolopyrimidine sulfamates class is expected to identify compounds with reduced activity against human Atg7, and thus even higher specificity for plasmodium. We note that the HIAQ and KMSKS motifs are conserved across apicomplexan and kinetoplastid parasites but not in metazoan organisms ( fig. S17). This suggests that ML901-like compounds could exhibit cross-pathogen inhibitory activity. Use of the sulfamates in a drug combination could prevent evolution of resistant mutants.
Our finding that nucleoside sulfamates can hijack Class I and Class II tRNA aaRSs, as well as E1s, opens up the possibility of designing bespoke small molecular weight, membrane permeable AFE inhibitors with adjustable specificity. In addition to charging tRNA and activating ubiquitin, AFEs are involved in activating fatty acids for degradation, biosynthesis of natural products, and other diverse pathways (21). Thus, nucleoside sulfamates may find applications in a broad range of infectious, metabolic and neurodegenerative diseases.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.